Introduction: IVD degeneration (IDD) is characterized by apoptosis, inflammation, structural deterioration, and extracellular matrix (ECM) degradation¹. The phosphatase pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) isozymes, PHLPP1 and PHLPP2 both promote apoptosis and inactivate AKT, which is a major regulatory upstream of transcription factor FOXO1. FOXO1 has been recently demonstrated to be essential to maintain IVD health in mice². The aim of this study was to identify if pharmacological inhibition of PHLPP activity can promote IVD health by mitigating pro-inflammatory and catabolic responses via FOXO1 in the nucleus pulposus (NP) of degenerating IVDs.

Methods: Human degenerated NP cells (n=3, Pfirrmann grade 4-5) were extracted following IRB approval. After reaching 85% confluency, NP cells (passage 1-2) were serum deprived (0.2% FBS) for 2 hours, followed by PHLPP Inhibitor treatment (25, 50 & 100mM of the small molecule PHLPP inhibitor NSC45586) for 30 minutes (immunoblotting for AKT and ERK phosphorylation and FOXO1 expression) or 24 hours (cell viability (MTT) and gene expression for KRT19, ACAN, MMP13, IL6). To investigate if PHLPP acts via FOXO1, a FOXO1 inhibitor (200nM, small molecule FOXO1 inhibitor AS1842856) was added to the culture media 1 hour before PHLPP inhibitor (100mM) treatment. Data were analyzed using Kruskal-Wallis-Testing (Graphpad Prism 8) with a statistical significance of p<0.05.

Results: PHLPP Inhibitor treatment of degenerated human NP cells promoted cell viability in a dose dependent manner (Figure 1A). PHLPP inhibitor treatment further increased gene expression of KRT19 and ACAN, while it decreased MMP13 and IL6(Figure 1B). Immunoblotting for AKT, ERK, as well as FOXO1 demonstrated an inhibitor dependent increase in AKT phosphorylation and FOXO1 deposition but a decrease in ERK phosphorylation (Figure 1C+D). Increased FOXO1 expression induced by the PHLPP inhibitor was further confirmed by immunofluorescent staining (Figure 1E). Combination of the inhibitors for PHLPP and FOXO1 demonstrated that inhibition of FOXO1 reversed the effects of PHLPP inhibition on FOXO1 protein expression (Figure 1E) and gene expression of KRT19, ACAN and MMP13 (Figure 1F).

Discussion: Our study demonstrated that the small molecule PHLPP inhibitor NSC45586 promoted NP cell health in degenerated human NP cells. PHLPP inhibition increased ECM synthesis and decreased pro-inflammatory responses. In degenerated human NP cells, the inhibitor promoted FOXO1 deposition and AKT phosphorylation while decreasing ERK phosphorylation. Activated FOXO1 and AKT and inactivated ERK play important roles in maintaining IVD health^2,3. These findings were somewhat surprising because it is known that AKT signaling inactivates FOXO1. It’s possible that PHLPP regulates FOXO1 expression independent of AKT. Our findings are also supported by others demonstrating evidence of AKT-independent FOXO1 in hematopoietic stem and progenitor cells⁴. Notably, inhibition of FOXO1 reversed the protective effects of PHLPP inhibitor on IDD, warranting further investigation in the correlation of PHLPP and FOXO1.

Acknowledgement: Funded by NIH R21 AR072222 and the Department of Orthopaedics at Emory University.

Reference: 1. Wuertz+ Eur Cell Mater 2012. 2. Alvarez-Garcia+ Aging cell 2018. 3.Zhou+ Connect. Tissue Res. 2020. 4. Liang+ Cell cycle. 2013.